弓形蟲(chóng)基因疫苗pBudCE4.1-AMA1-ROP18免疫小鼠的保護(hù)作用研究
發(fā)布時(shí)間：2018-12-23 07:58
【摘要】：弓形蟲(chóng)為寄生于細(xì)胞內(nèi)的原蟲(chóng),它感染所有的恒溫動(dòng)物,包括人類,是寄生于人體最普遍的寄生蟲(chóng)之一,全球約有三分之一的人口感染弓形蟲(chóng)。弓形蟲(chóng)感染在先天感染和免疫系統(tǒng)受損的個(gè)體中有高發(fā)病率和高死亡率。先天感染可導(dǎo)致流產(chǎn)、新生兒畸形或胎兒出生后導(dǎo)致其他缺陷,如失明和嚴(yán)重的認(rèn)知缺陷等。在免疫系統(tǒng)受損的個(gè)體中,感染寄生蟲(chóng)可導(dǎo)致腦炎和神經(jīng)損傷。除此之外,食用感染弓形蟲(chóng)包囊動(dòng)物的肉,是人體感染弓形蟲(chóng)的一個(gè)主要途徑。 棒狀體蛋白18(ROP18)為弓形蟲(chóng)棒狀體蛋白,當(dāng)弓形蟲(chóng)進(jìn)入宿主細(xì)胞時(shí),會(huì)生成一層保護(hù)膜包裹自身,使自己受細(xì)胞環(huán)境影響而不被殺死,ROP18的作用則是讓宿主細(xì)胞某些蛋白失效而無(wú)法破壞這層保護(hù)膜。其作用機(jī)制是通過(guò)和宿主細(xì)胞中一類與免疫有關(guān)的鳥(niǎo)苷酸酶(GTPase)相結(jié)合,使得后者失效,進(jìn)而無(wú)法破壞弓形蟲(chóng)周圍的保護(hù)膜。AMA1為弓形蟲(chóng)頂端膜抗原,是一種微線體蛋白,為I型膜蛋白,分泌于弓形蟲(chóng)表面,在所有復(fù)頂亞門(mén)寄生蟲(chóng)中高度保守。有實(shí)驗(yàn)表明,AMAl在弓形蟲(chóng)入侵宿主細(xì)胞的過(guò)程中起重要作用,AMA1缺失將使弓形蟲(chóng)無(wú)法進(jìn)入宿主細(xì)胞。 pBudCE4.1表達(dá)載體可同時(shí)表達(dá)哺乳動(dòng)物細(xì)胞系的兩個(gè)基因,并可同時(shí)獨(dú)立表達(dá)兩個(gè)重組蛋白,可在多種類型哺乳動(dòng)物細(xì)胞中高效穩(wěn)定的表達(dá)。 本研究根據(jù)已發(fā)表的AMA1mRNA序列和ROP18DNA序列,設(shè)計(jì)合成引物,構(gòu)建真核表達(dá)質(zhì)粒pBudCE4.1-AMA1、pBudCE4.1-ROP18,然后構(gòu)建pBudCE4.1-AMA1-ROP18真核重組質(zhì)粒。然后轉(zhuǎn)染人包皮成纖維細(xì)胞(HFF),48-72小時(shí)后,提取細(xì)胞RNA,通過(guò)SDS-PAGE及逆轉(zhuǎn)錄鑒定其表達(dá)情況,證實(shí)重組質(zhì)粒在HFF細(xì)胞中正常表達(dá)。此后,通過(guò)肌肉注射重組質(zhì)粒免疫BALB/c小鼠,設(shè)立PBS對(duì)照組及空載體對(duì)照組,于第15天和第30天加強(qiáng)免疫,并在免疫前取血,收集血清；在第56天時(shí)進(jìn)行攻擊實(shí)驗(yàn)。將收集的血清進(jìn)行免疫學(xué)檢測(cè),檢測(cè)結(jié)果表明重組表達(dá)載體誘發(fā)宿主的免疫應(yīng)答反應(yīng)效果良好。通過(guò)攻擊感染實(shí)驗(yàn)的驗(yàn)證,證明重組DNA疫苗對(duì)小鼠均具有一定的免疫保護(hù)作用,而且,重組質(zhì)�；蛞呙绲拿庖咝Чh(yuǎn)遠(yuǎn)比單基因疫苗的免疫效果高,這對(duì)今后進(jìn)一步研制弓形蟲(chóng)基因疫苗奠定了堅(jiān)實(shí)的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is one of the most common parasites in human body. It infects all isothermal animals including humans. About 1/3 people worldwide are infected with Toxoplasma gondii. Toxoplasma gondii infection is associated with high morbidity and mortality in individuals with congenital infections and impaired immune systems. Congenital infections can lead to miscarriage, neonatal malformations, or other defects after birth, such as blindness and severe cognitive impairment. In individuals with impaired immune systems, infection with parasites can lead to encephalitis and nerve damage. In addition, eating the meat of Toxoplasma gondii cyst animals is a main way to infect Toxoplasma gondii. Coryloid protein 18 (ROP18) is a Toxoplasma gondii rodlike protein. When Toxoplasma gondii enters the host cell, it forms a protective membrane that wraps itself in such a way that it is not killed by the cellular environment. The effect of ROP18 is to make certain proteins of host cells fail to destroy the protective membrane. The mechanism is that by combining with a class of immune-related guanylidase (GTPase) in host cells, the latter fails to destroy the protective membrane around Toxoplasma gondii. AMA1 is a microline protein, which is the top membrane antigen of Toxoplasma gondii. It is a type I membrane protein secreted on the surface of Toxoplasma gondii and highly conserved in all parasites. Some experiments have shown that AMAl plays an important role in the process of Toxoplasma gondii invading host cells, and AMA1 deletion will prevent Toxoplasma gondii from entering host cells. PBudCE4.1 expression vector can express two genes of mammalian cell line simultaneously and can express two recombinant proteins independently at the same time. It can be expressed efficiently and stably in many kinds of mammalian cells. Based on the published AMA1mRNA sequence and ROP18DNA sequence, primers were designed and synthesized to construct the eukaryotic expression plasmid pBudCE4.1-AMA1,pBudCE4.1-ROP18, and then construct the pBudCE4.1-AMA1-ROP18 eukaryotic recombinant plasmid. After transfection of human prepuce fibroblasts (HFF),) for 48 to 72 hours, RNA, was extracted and its expression was identified by SDS-PAGE and reverse transcription. The normal expression of the recombinant plasmid was confirmed in HFF cells. After that, BALB/c mice were immunized by intramuscular injection of recombinant plasmid, and PBS control group and empty vector control group were set up. The mice were immunized on the 15th and 30th days, blood was collected before immunization, and serum was collected, and the attack test was carried out on the 56th day. The collected serum was detected by immunology, and the results showed that the recombinant expression vector could induce the host immune response. The results showed that the recombinant DNA vaccine had a certain immune protective effect on mice, and the immune effect of recombinant plasmid gene vaccine was much higher than that of single gene vaccine. This laid a solid experimental foundation for the further development of Toxoplasma gondii gene vaccine.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
本文編號(hào)：2389656
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is one of the most common parasites in human body. It infects all isothermal animals including humans. About 1/3 people worldwide are infected with Toxoplasma gondii. Toxoplasma gondii infection is associated with high morbidity and mortality in individuals with congenital infections and impaired immune systems. Congenital infections can lead to miscarriage, neonatal malformations, or other defects after birth, such as blindness and severe cognitive impairment. In individuals with impaired immune systems, infection with parasites can lead to encephalitis and nerve damage. In addition, eating the meat of Toxoplasma gondii cyst animals is a main way to infect Toxoplasma gondii. Coryloid protein 18 (ROP18) is a Toxoplasma gondii rodlike protein. When Toxoplasma gondii enters the host cell, it forms a protective membrane that wraps itself in such a way that it is not killed by the cellular environment. The effect of ROP18 is to make certain proteins of host cells fail to destroy the protective membrane. The mechanism is that by combining with a class of immune-related guanylidase (GTPase) in host cells, the latter fails to destroy the protective membrane around Toxoplasma gondii. AMA1 is a microline protein, which is the top membrane antigen of Toxoplasma gondii. It is a type I membrane protein secreted on the surface of Toxoplasma gondii and highly conserved in all parasites. Some experiments have shown that AMAl plays an important role in the process of Toxoplasma gondii invading host cells, and AMA1 deletion will prevent Toxoplasma gondii from entering host cells. PBudCE4.1 expression vector can express two genes of mammalian cell line simultaneously and can express two recombinant proteins independently at the same time. It can be expressed efficiently and stably in many kinds of mammalian cells. Based on the published AMA1mRNA sequence and ROP18DNA sequence, primers were designed and synthesized to construct the eukaryotic expression plasmid pBudCE4.1-AMA1,pBudCE4.1-ROP18, and then construct the pBudCE4.1-AMA1-ROP18 eukaryotic recombinant plasmid. After transfection of human prepuce fibroblasts (HFF),) for 48 to 72 hours, RNA, was extracted and its expression was identified by SDS-PAGE and reverse transcription. The normal expression of the recombinant plasmid was confirmed in HFF cells. After that, BALB/c mice were immunized by intramuscular injection of recombinant plasmid, and PBS control group and empty vector control group were set up. The mice were immunized on the 15th and 30th days, blood was collected before immunization, and serum was collected, and the attack test was carried out on the 56th day. The collected serum was detected by immunology, and the results showed that the recombinant expression vector could induce the host immune response. The results showed that the recombinant DNA vaccine had a certain immune protective effect on mice, and the immune effect of recombinant plasmid gene vaccine was much higher than that of single gene vaccine. This laid a solid experimental foundation for the further development of Toxoplasma gondii gene vaccine.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 金春梅;張厚雙;薛書(shū)江;權(quán)根花;崔金偉;王興貴;;弓形蟲(chóng)AMA1蛋白真核表達(dá)載體的構(gòu)建及免疫活性檢測(cè)[J];安徽農(nóng)業(yè)科學(xué);2010年29期
2 程相朝,張敏,王建軍,王婷;DNA疫苗的主要意義與特點(diǎn)[J];河南科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年03期
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